The DNA mismatch repair (MMR) system corrects DNA synthesis errors that occur during replication and also is involved in several other DNA transactions. MMR is initiated by MutS and MutL homologs, which are highly conserved throughout prokaryotes and eukaryotes. They are both dimers and contain DNA binding and ATPase activities that are essential for MMR in vivo. Inactivation of these proteins leads to increased mutagenesis, improper recombination, and resistance to the cytotoxic effects of several DNA damaging agents. In humans, mutations in the mismatch repair genes are directly linked to hereditary non-polyposis colorectal cancer (HNPCC) and are associated with several sporadic cancers. Because of the diversity of functions carried out by the MMR proteins, it will be essential to understand the molecular mechanisms that underlie these different processes to develop effective treatment for the associated diseases and cancers. In eukaryotes, MutS? (MSH2-MSH6) and MutL? (MLH1-PMS2) are the primary MutS and MutL homologs responsible for initiation of MMR. MutS? initiates repair by binding to a mismatch and undergoing an ATP-dependent conformational change that promotes its interaction with MutL?. PCNA then activates MutL? to incise the daughter strand both 5' and 3' to the mismatch. Subsequently, MutS? activates the 5'-3' exonuclease EXO1 to processively excise the DNA containing the incorrect nucleotide. Finally, DNA polymerase ? or ? catalyzes resynthesis, and DNA ligase seals the nick. Structural and biochemical studies, including several from our lab, indicate that the conformational dynamics and assembly states of the proteins and protein-DNA complexes are central to the regulation of MMR. The overall goal of this proposal is to elucidate the structure-function relationships that govern the initiation steps of MMR. We propose a systematic series of experiments, in which we characterize the structural, conformational, and dynamic properties of MMR complexes formed with MutS? using atomic force microscopy (AFM), our newly developed Dual Resonance frequency Enhanced Electrostatic force Microscopy (DREEM), which allows visualization of the path of the DNA through the proteins, and single-molecule fluorescence. These studies will be complemented with a thorough examination of the biochemical and functional properties done by our collaborators in Paul Modrich's and Peggy Hsieh's laboratories. This combination of techniques will allow us to fully characterize the binding, dynamic, conformational, and functional properties of complexes that govern MMR and the preservation of genomic stability. Our goals are to: 1) dissect the molecular mechanisms of mismatch recognition by MutS?, and 2) determine the conformational properties of MutS?-MutL?-mismatch complexes that govern the initiation of repair.